水稻雄性不育基因OsPL1在花药发育中的机制解析

 2023-08-08 07:08

论文总字数:29821字

摘 要

我国水稻育种至今经历了三次重大革命,水稻雄性不育系的应用和研究始终具有广阔前景,该方面理论的丰富与发展更是对分子育种具有重要意义。在本论文中,水稻雄性不育基因OsPL1材料是通过60Coγ射线辐射诱变籼稻品种3037得到,利用免疫荧光染色分析、qRT-PCR扩增、互补载体构建等技术,经过表型鉴定、遗传分析、基因定位和结构分析、花药半薄树脂切片分析、花粉母细胞减数分裂进程分析、蛋白表达鉴定等过程,最终得出结论:OsPL1基因为一隐性单核基因突变,定位在水稻10号染色体上,突变后导致该基因的第6外显子内基因序列由TTCCGC突变成GTCCCTC,且突变体中出现AvaII酶酶切位点,可用于后期基因型酶切鉴定。其基因在穗中的花药组织中表达量最高。此外,OsPL1基因的突变并不影响雄配子减数分裂的进程,但延迟了绒毡层的凋亡进程,使得花粉发育异常无花粉释放,由此造成水稻雄性不育。

关键词:水稻(Oryza sativa),雄性不育,OsPL1基因,花药发育,绒毡层

Abstract:After three major revolutions in rice breeding, the application and research of rice male sterile still has a broad prospect, especially the rich and the development in its theory is of great significance for the molecular breeding. In this study, the OsPL1 mutant material is obtained by 60Coγ irradiation induced the mutation of an indica rice variety 3037. Using the immunofluorescence staining analysis, qRT-PCR amplification and complementary alalysis, , together with the phenotypic identification, genetic analysis, gene localization and structural analysis, anther semi-thin resin section analysis, pollen mother cell meiosis process analysis, protein expression identification and other processes, we concluded that the OsPL1 mutation, as a recessive mononuclear gene mutations, locates on the rice chromosome 10, and its mutation occurs on the 6th exon of the gene, thus lead to the original gene sequence TTCCGC into the GTCCCTC. Moreover, this mutation can be cut with the restriction endonuclease enzyme Avall , which is used to identify the genetype of OsPL1. The expression of OsPL1 gene are mainly distributed in the plasma membrane, and had the highest expression in anther tissue; The mutation of OsPL1 gene did not affect the process of the meiosis of male gametes, but delayed the transfer of the tapetum, causing the abnormal pollen development , and finally resulting in the rice male sterility.

Keywords:rice (Oryza sativa), male sterility, OsPL1, anther development, tapetum

目 录

1 引言 3

1.1 水稻雄性生殖的发育过程 3

1.1.1 水稻雄蕊及花药的发育 3

1.1.2 花药壁绒毡层的发育过程 5

1.2 水稻雄性不育研究进展 6

1.2.1 水稻雄性不育系的分类 6

1.2.2 水稻雄性不育的不育机制 6

1.2.3 研究思路和技术手段 8

1.3 本研究的目的和意义 9

2 材料与方法 9

2.1 实验材料 9

2.1.1 水稻材料 9

2.1.2 实验试剂及仪器 9

2.2 实验方法 10

2.2.1 材料种植与实验群体构建 10

2.2.2 统计学分析确定遗传类型 10

2.2.3 KI-I2染色观察花粉 10

2.2.4 水稻总DNA的提取与基因结构分析 11

2.2.5 水稻组织RNA的提取与表达量分析 11

2.2.6 载体构建及水稻植株转化 13

2.2.7 水稻花药半薄树脂切片 13

2.2.8 特异蛋白的免疫荧光染色分析 14

2.2.9 测序及数据来源 14

3 结果与分析 14

3.1 OsPL1突变体的表型鉴定 14

3.2 OsPL1突变体遗传分析 15

3.3 OsPL1基因结构与突变位点鉴定分析 15

3.4 OsPL1突变体的互补验证 16

3.5 花药半薄树脂切片分析 17

3.6 花粉母细胞染色体表型分析 17

3.7 花粉母细胞染色体免疫荧光染色分析 18

3.8 OsPL1基因功能缺陷对绒毡层发育相关基因的表达量影响 18

结 论 20

参考文献 21

在校期间已获成果 26

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